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The bioinformatic person is doing all that. She trimmed the adapters and tried aligning the reads to the genome using tophat. She got 40% alignment there. We tried blasting some of the unaligned reads and realized that something went wrong with the tophat run as some of them were aligning to chromosome M, chr 1, 4 etc. She will be doing the alignment again with STAR this time but to save on time she also ran the RSEM along side and got these low percentage alignments to transcriptome so I wanted to know if we are missing out on anything?
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It would be helpful if you posted some basic quality metrics, such as you get from FastQC. There's not enough information to determine what the problem is or even if there is a problem.Comment
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Ok. I have looked at the FastQC report of the samples and it looks fine to me. What kind of information should I give from FastQC report?Comment
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The whole thing as PDF is good way to start. But particularly the quality histograms, anything that failed the tests, and anything related to mapping. And the stats from adapter trimming, the command lines you used for each program, the top hits and mapping rate you got from Blast... etc.Comment
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I will have to ask my bioinformatician for the stats after adapter trimming. I will get back on this. Thanks for your time.Comment
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Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.07-01-2026, 11:43 AM
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I’m not a sequencing expert. I’m a purification scientist who uses NGS to evaluate workflows my group develops. With this perspective, we think about the sample first and the NGS workflow second. The sequencer is an exceptionally honest reporter, but it can only report on what you give it, so whether you get clean, interpretable data from an NGS workflow is largely determined before you begin.
Here are nine questions we think about, in roughly the order they matter, before...
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